Functions and Activities of p53 and Mdm2 in Normal and Cancer Cells. The p53 protein, a major suppressor of cancer in animals and humans, is a DNA sequence specific regulator of transcription of hundreds of genes whose products mediate cellular outcomes such as cell cycle arrest, cell death, senescence, metabolic changes, DNA repair and others that are consistent with tumor suppression. While a potent regulator of such processes in cells undergoing various forms of genotoxic or oncogenic stress, if allowed to function unchecked, p53 can be damaging or even lethal to cells and tissues. Keeping p53 under tight control is therefore as important as allowing it to function when needed. The two proteins that play the most central role in suppressing p53 are Mdm2 and its homologue MdmX. Over half of all human malignancies (depending on the tumor type) harbor wild-type p53, which if released from Mdm2/X, might be effective as a cytostatic or cytotoxic anti-tumor agent. Yet reagents (peptides and small molecules) that prevent Mdm2 from binding to p53 have not yet been fully realized as effective therapeutic agents. Similarly, there few effective drugs in the clinic that prevent the oncogenic activities of mutant p53. Further, increasing lines of evidence document p53-independent roles of Mdm2 and MdmX in cancer cells that can be exploited for therapeutic and diagnostic purposes. The work proposed in this application for an Outstanding Investigator Award follows a long history of contributions in the laboratory of the PI to our understanding p53, Mdm2 and MdmX that continue to the present day. The plans going forward build on new findings in the PI's lab, and incorporate novel technologies in order to gain more global and mechanistic information about the p53/Mdm axis. The research proposed will extend recent discoveries related to (i) a novel mechanism by which Mdm2 degrades p53 and the possibility that this finding can lead to new therapeutics that prevent Mdm2 degradation of wild-type p53 or conversely facilitate Mdm2 degradation of oncogenic mutant forms of p53, (ii) a series of CRISPR-derived cell lines harboring unique Li-Fraumeni-Syndrome p53 mutations; (iii) p53- independent roles of Mdm2 and MdmX that may either prevent or support oncogenesis, (iv) long non-coding RNAs that are p53 targets and which may provide new biomarkers in human cancers; (v) new approaches to understand how p53 identifies its sites in vitro and in vivo and, (vi) examination at single molecule resolution the localization of wild-type and mutant forms of p53 and Mdm2 after different stimuli. The long term goals of this program will hopefully lead to new tools for diagnosis and treatment of sporadic and inherited cancers.